TREM2 expression promotes liver and peritoneal M2 macrophage polarization in mice infected with Schistosoma japonicum

Abstract Schistosomiasis is a tropical parasitic disease that damages the liver and poses a serious threat to human health. Macrophages play a key role in the development of liver granulomas and fibrosis by undergoing polarization from M1 to M2 type during schistosomiasis. Therefore, regulating macrophage polarization is important for controlling pathological changes that occur during this disease. Triggering receptor expressed on myeloid cells 2 (TREM2) expressed on the surface of macrophages, dendritic cells and other immune cells has been shown to play a role in inhibiting inflammatory responses and regulating M2 macrophage polarization, however its role in macrophage polarization in schistosomiasis has not been investigated. In this study, we confirmed that TREM2 expression was upregulated in the livers and peritoneal macrophages of mice infected with Schistosoma japonicum. Moreover, the TREM2 expression trend correlated with the expression of M2 macrophage polarization‐related molecules in the liver tissues of S. japonicum‐infected mice. Using Trem2 −/− mice, we also showed that Trem2 deletion inhibited Arg1 and Ym1 expression in liver tissues. Trem2 deletion also increased the number of F4/80 + CD86+ cells in peritoneal macrophages of infected mice. In summary, our study suggests that TREM2 may be involved in M2 macrophage polarization during schistosomiasis.

other immune cells has been shown to play a role in inhibiting inflammatory responses and regulating M2 macrophage polarization, however its role in macrophage polarization in schistosomiasis has not been investigated. In this study, we confirmed that TREM2 expression was upregulated in the livers and peritoneal macrophages of mice infected with Schistosoma japonicum. Moreover, the TREM2 expression trend correlated with the expression of M2 macrophage polarization-related molecules in the liver tissues of S. japonicum-infected mice. Using Trem2 −/− mice, we also showed that Trem2 deletion inhibited Arg1 and Ym1 expression in liver tissues. Trem2 deletion also increased the number of F4/80 + CD86+ cells in peritoneal macrophages of infected mice. In summary, our study suggests that TREM2 may be involved in M2 macrophage polarization during schistosomiasis.

K E Y W O R D S
macrophage, polarization, Schistosoma japonicum, triggering receptor expressed on myeloid cells 2 macrophages promote the release of pro-inflammatory factors such as tumour necrosis factor α (TNFα), interleukin 12 (IL-12) and interleukin 1 (IL-1), leading to an inflammatory response that induces acute schistosomiasis and liver damage. 5 Subsequently, macrophages tend to polarize into alternatively activated macrophages (ACMs, M2-type) following stimulation by SEA. 3,4 M2 macrophages promote the production of anti-inflammatory factors, including IL-10, IL-4 and transforming growth factor β (TGFβ), which help to inhibit the inflammatory response during the acute infection phase, resulting in the transition from acute to chronic schistosomiasis. 6 Suppression of the expression levels of these anti-inflammatory factors may contribute to the high production of reactive oxygen and nitrogen intermediates and the high expression of nitric oxide synthase 2 (NOS2) in the livers from mice infected with Schistosoma mansoni, resulting in a large number of deaths in the acute infection phase of mice. 6,7 Therefore, the shift from M1 to M2 macrophages plays an important role in the formation of granulomas and fibrosis. 8,9 However, the underlying mechanism of this macrophage phenotypic transformation remains unclear.
Previous studies have shown that multiple cytokines and receptors are involved in the process of macrophage polarization. 4,9,10 Among them, NOS2, IL-1β and TNFα are highly expressed in M1 macrophages, while arginase 1 (ARG1), mannose receptor C-type 1 (MRC1, CD206), and chitinase like 3 (CHIL3, YM1) are expressed in M2 macrophages. 11,12 In schistosomiasis, Ye et al. 11 reported that both Arg1 and Ym1 expression levels were increased in livers and in peritoneal macrophages from mice infected with Schistosoma japonicum for 8 weeks. Zhu et al. 13 also demonstrated that the dynamic expression levels of Nos2 and Arg1 mRNA in peritoneal macrophages from mice infected with S. japonicum for various time points indicated the shift of macrophages from M1 to M2 type in schistosomiasis.
Triggering receptors expressed on myeloid cells (TREMs), including TREM1, TREM2 and TREM3, have garnered attention from researchers in recent years. 14

| RNA extraction and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR)
RNA was extracted from tissue homogenate in Trizol and used as a template for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The cDNA was then applied as the template for qPCR which was performed using the TB Green Premix Ex Taq II Kit from TAKARA (Japan). Primer sequences are shown in Table 2.

| Western blotting
Protein was extracted from tissues using RIPA buffer (Beyotime) and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The samples were then transferred to polyvinylidene fluoride membranes (Merck) and incubated with 5% nonfat milk. After blocking and washing, the samples were incubated with primary antibody (AF1729, R&D) at 4°C overnight followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech) at room temperature for 2 h. The ECL kit (Merck) was used for visualization on a chemiluminescent instrument.

| Immunofluorescence staining and immunohistochemistry
The obtained liver tissues were immersed in formalin solution for at least 24 h, then paraffin embedded in a multifunctional embedding machine. The tissues were then sliced in a rotary microtome.
For immunofluorescence staining, the tissue sections were dewaxed using xylene and placed in citrate buffer for antigen repair.
After endogenous peroxidase was blocked, the tissue sections were incubated with donkey serum followed by an antibody against TREM2 (ab86491, Abcam) and F4/80 (30,325 T, CST). Next, the sections were incubated with the associated secondary antibody (112-605-062 and 711-545-152, Jackson) and DAPI. After being sealed with 50% glycerin, the samples were observed and photographed via microscopy (Leica).
For immunohistochemistry experiments, the previously de-

| Flow cytometry
Peritoneal macrophages were obtained from mice 12

| Statistical analysis
All data are presented as mean ± SEM of at least three independent experiments and analysed in SPSS 20.0 using One-way ANOVA or Independent Samples T-tests. p < 0.05 was considered statistically significant between two groups.

| TREM2 expression is upregulated in livers of mice infected with Schistosoma japonicum
We first measured TREM2 expression in the livers of S. japonicuminfected mice and found that Trem2 mRNA expression was significantly upregulated at 9 and 12 weeks post-infection compared to uninfected controls (**p < 0.01, Figure 1A). TREM2 upregulation in TA B L E 1 Primers for genotype identification.

Primer names Sequences
Trem2-5S-in-tF1 a ATGCC TGT CTC CCA AGA ACAGA These primers were paired and the PCR products indicated Trem2 deletion in mice. a,c These primers were paired and the PCR products indicated wild-type mice. infected mice was also observed at the protein level via western blotting ( Figure 1B). Using immunofluorescence, we also found that the numbers of both F4/80+ and TREM2+ cells were higher in the livers of S. japonicum-infected mice than those of controls ( Figure 1C). Notably, F4/80+ TREM2+ cells were also elevated and observed surrounding the egg granuloma in infected livers ( Figure 1C). These data suggest that S. japonicum infection induces TREM2 expression in mouse livers.

| Arg1 and Ym1 mRNA expression is enhanced in the liver tissues of mice infected with Schistosoma japonicum
Since TREM2 was found to be expressed in F4/80+ cells, we next
Immunohistochemistry analysis revealed that TREM2 expression was upregulated in livers from wild-type mice infected with S. japonicum after 6 weeks or 12 weeks, as evidenced by an increase in the

| Trem2 deletion increases the number of F4/80 + CD86+ cells in peritoneal macrophages
We measured the expression of TREM2 in peritoneal macrophages from mice infected with S. japonicum and found that the percentage of TREM2 positive F4/80 + CD11b + cells was enhanced at 6 weeks (p < 0.05), 9 weeks (p < 0.01) and 12 weeks (p < 0.001) post-infection relative to uninfected controls ( Figure 4A). This indicates that TREM2 expression is upregulated in peritoneal macrophages during

S. japonicum infection in mice.
Primary macrophages from wild-type mice can be polar- Previous studies have highlighted the potential role of TREM2 in the development and progression of liver diseases. Specifically, upregulation of TREM2 expression has been observed in both carbon tetrachloride-induced acute and chronic liver injury models, in liver tissues of mice following biliary ligation, 30 as well as in liver tissues of patients with cirrhosis and hepatocellular carcinoma (HCC). 30,31 In this study, we found that TREM2 expression was also increased in the livers of S. japonicum-infected mice. Interestingly, TREM2+ cells mainly F I G U R E 2 The relative expression of Arg1 and Ym1 mRNA is enhanced in the liver tissues of Schistosoma japonicuminfected mice. Arg1 (A), Ym1 (B), Nos2 (C) and Il1β (D) mRNA expression levels in livers were detected by RT-qPCR and compared to expression in uninfected controls. *p < 0.05, **p < 0.01. those reported by Jiawei Zhang et al. 15 and Xiaobao Zhang et al. 34 They found that overexpression of TREM2 could promote Arg1 mRNA expression in microglia and suppress LPS-induced elevation of TNFα and IL-1β in microglia. 34 TREM2 may regulate the balance of M1/M2 polarization in microglia induced by curcumin or LPS. 15,34 Collectively, these results suggest that TREM2 may be involved in M2 macrophage polarization in schistosomiasis.

F I G U R E 3 Trem2 deletion inhibits
In conclusion, S. japonicum-infection may induce TREM2 expression in mouse livers and TREM2 may be involved in M2 macrophage polarization during schistosomiasis. However, additional research is required in order to determine the mechanism.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have stated explicitly that there are no conflicts of interest in connection with this article.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the conclusions of this article are included within the article